FLAVONOID AND BIFLAVONOID DERIVATIVES, THEIR PHARMACEUTICAL COMPOSITIONS, THEIR ANXIOLYTIC ACTIVITY

Abstract: Certain flavonoids, notably derivatives of flavone, chrysin and apigenin together with dimers thereof such as amentoflavone, have been found to possess anxiolytic properties (i.e. anxiety reducing properties) without exhibiting a sedative effect. The compounds are defined by general formula (I), wherein R<1>, R<2>, R<3>, R<4>, R<5> and R<8> are independently selected from H, OH, R, NO2, halo, OR, NH2, NHR, NR2, COOR, COOH, CN or a sugar group; R<6>, and R<7> are both H, or R<6> and R<7> together form a single bond; R is C1-6 alkyl or alkenyl; or the administration of an effective non-toxic amount of a biflavonoid which is a dimer of a compound of general formula (I). Novel compounds and pharmaceutical formulations are also described.

Description: FLAVONOID AND BIFUWONOID DERIVATIVES, THEIR PHARMACEUTICAL COMPOSITIONS, THEIR ANXIOLYTIC ACTIVITY The present invention relates to certain flavonoids which have been found to have anxiolytic properties (i.e. anxiety reducing) without corresponding depression of the central nervous system which is commonly also found in known sedatives such as benzodiazepines. Some compounds of the invention are novel; other compounds are known but no pharmaceutical uses have previously been described. Flavone is a known compound which is described in the Merck Index (entry 4030) . Chrysin (2261) and apigenin (763) are other known flavonoids. Chrysin has been described as having binding properties for benzodiazepine receptors and anticonvulsant properties in Medina J.H. et al. Biochem. Pharmacol; 40: 2227-2232, 1990. This reference also suggests that chrysin may possess myorelaxant (i.e. muscle relaxant) action. Flavone, 2-phenyl-4H-l-benzopyran-4-one has the 2>' formula / . o Chrysin is 5 , 7-dihydroxyflavone Apigenin is 4 ' , 5 , 7- trihydroxyflavone . The present invention relates to a method of treatment of anxiety in a patient which comprises administering to the patient an effective non-toxic amount of a flavonoid compound of general formula (I) : where R 1 , R 2 , R 3 and R 4 , R 5 and R 8 are independently selected from H, OH, R, N0 2 , halo, OR, NH 2 , NHR, NR 2 , COOR, COOH, CN, or a sugar group; R 6 and R 7 are both H, or R 6 and R 7 together form a single bond; R is Cι_ 6 alkyl or alkenyl; or the administration of an effective non-toxic amount of a biflavonoid which is a dimer of a compound of general formula (I) and wherein R 1 to R 8 and R have the meanings given for general formula (I) . It is found that the compounds of the present invention have anxiolytic properties without the associated depression of the central nervous system (e.g. sedative and muscle relaxant effects) commonly found with benzodiazepines. This may allow patients to be treated for anxiety without inducing sedative or myorelaxant side-effects. It is found further that compounds of the present invention may not show an anti-convulsant activity commonly found with benzodiazepines. The compounds of general formula (I) wherein R 1 , R 2 , R 3 , R 4 and R 5 may independently be H, OH or halo (including F, Cl, Br or I) are preferred. The preferred halo substituent is Br, F or Cl. The following compounds of general formula (I) are particularly preferred wherein R 1 , R 3 , and R 5 may be hydroxy. Also wherein R 1 , and R 3 are hydroxy and R 5 is halo. Alternatively wherein R 3 is hydroxy and R is hydrogen, or wherein R 3 and R 1 are both hydroxy is preferred. More preferably R 2 and R 4 are both halo. More preferably R 5 is OH or halo. When R 5 is halo it is particularly preferred that the compounds of general formula (I) are substituted at the 2 ' position. The compounds of general formula (I) which are flavone, chrysin, apigenin and the derivatives 2'-chlorochrysin, 2'-fluorochrysin, 6,8-dibromochrysin and 7-bromoflavone are particularly preferred. Compounds where R ό and R 7 together form a single bond are flavone derivatives, whereas compounds where R 6 and R 7 are both H are flavonone derivatives. The sugar group may be any of the known sugars, including onosaccharides, disaccharides and polysaccharides; and may in particular be glycosyl, galactopyranosyl or annopyranosyl. The biflavonoid is a dimer of two covalently bonded moieties which are each of general formula (I) as set out above. Bonding between the two moieties generally occurs at the 3'-position of one moiety and the 8-position of the other moiety. The preferred biflavonoid has general formula (II) wherein R 1 to R 8 and R have the same meanings as for general formula (I) . The compounds of general formula (II) wherein R 1 , R 3 and R 5 in each of the dimer moieties of general formula (I) are hydroxy or methoxy are preferred. The compounds of general formula (II) wherein the compounds are amentoflavone, ginkgetin or isoginkgetin are preferred. Pharmaceutical formulations include at least one compound of general formula (I) or (II) together with at least one pharmaceutically acceptable carrier or excipient. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. It should be understood that the flavonoid compounds of the present invention can be administered in the form of pharmaceutically acceptable salts or esters thereof. Salts are usually acid addition salts (e.g. with hydrohalogen acids) or acceptable metal salts (e.g. Na, Ca, Mg) . Formulations include those adapted for oral, rectal, nasal, vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Formulations of the present invention adapted for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethylcellulose) , lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethylcellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile. Formulations for rectal administration may bepresented as a suppository with a suitable base comprising for example cocoa butter or a salicylate. Formulation for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate. Formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. The dose will depend on a number of factors known to the skilled physician including the severity of the conditions, the identity of the recipient; and also the efficacy and toxicity cf the particular compound of general formula (I) which is being administered. Generally doses in the range 0.1-100 mg/kg body weight may be used, particularly 1-10 mg/kg. The frequency of administration will vary depending on the rate of metabolism or excretion of the administered compound, but may be repeated daily, optionally as two or more sub-doses. Unit doses of 20 to 500mg, preferably 100 to 400mg may be used. The present invention further relates to a flavonoid compound of wherein R 1 , R , R 3 , R 4 and R 5 are independently selected from H, OH and halo. R 6 and R 7 are both H, or R 6 and R 7 together form a single bond; and R is Cι_ 6 alkyl or alkenyl. Embodiments of the invention will now be described by way of example only. EXAMPLE 1 (Preparation of synthetic flavonoids) 1. Preparation of haloqenated chrysin 2 / -fluorochrysin and 2'-chlorochrysin were prepared by the Floc'h Lefeuvre synthesis (Tetrahedron Lett. 27, 5503-5504, 1985) by reaction of ortho-fluoro or chlorobenzoyl chlorides with the ylid obtained from 2,4,6-trihydroxyphenacylidene-triphenylphosphorane. Other compounds including 6,8 dibromo chrysin were prepared from SUBSTITUTE SHEET (RULE 2Q chrysin using the classical Allan-Robinson synthetic route (J. Allan and R. Robinson (1924) J. Chem. Soc. p2192) . « Preparation of 7-bromo flavone Bromine was added to a solution of flavanone in carbon tetrachloride at 0°C. The ratio of bromine/flavanone was 1.3 in molar terms. The temperature of the solution was raised to 30°C and kept there for 1 hour. The temperature of the solution was then raised to 65°C and kept there for 45 minutes. The reaction mixture was then extracted with an equal volume of a saturated solution of sodium metabisulphite and then dried with anhydrous sodium sulphate. The product was then recovered by evaporation of the solvent. This gave a mixture of brominated flavones, in which the only active ingredient was identified by NMR analysis as 7-bromo flavone (as shown in Figure 19) . EXAMPLE 2 (Experimental effect of flavonoids) Animals Male CF1 mice from our breeding stock weighing 28-35g were used. The animals were placed in groups of 10-12 with free access to water and food, and maintained on a 12h/12h day-night cycle. Drugs: Diazepam (DZ; Hoffman-La Roche) and the flavonoids were dissolved in DMSO 40%, NaOH 0.1N (7:3: v/v) at pH 8.2 Control animals were injected with the same vehicle (VEH) . RO 15-1788 (Hoffmann-La Roche) was suspended in DMSO 10%, propyleneglycol 10% in distilled water. Experimental Devices (a) Elevated Plus Maze: consisted of 4 perpendicularly disposed wood arms (20 x 5cm; two had 35cm high wood walls, and two were open) linked by a central 10 x 10 cm square. The maze was suspended 50cm from the room floor. Animals were placed on the central part of the maze facing a closed arm. This test has been widely validated to measure anxiety in rodents. The number of entries into and the time spent in the open and closed arms were counted during 5 min. A selective increase in the parameters corresponding to open arms reveals an anxiolytic effect. Total exploratory activity (number of entries in both arms) was also determined. (b) Holeboard Test: consisted of a wood box (60 x 60 x 30cm) with four 2cm diameter holes equidistant in the floor. The number of head-dips and the time spent head-dipping were counted during 5 min. An increase in the number and the time spent head-dipping implies a greater exploratory activity. A decrease of both parameters reveals a sedative behavior. (c) Locomotor Activity Test: we used an OPTO-VARIMEX apparatus consisting of a glass box (36 x 15 x 20cm) and two lateral bars with 15 light beams (0.32 cm diameter, beam spacing 2.65 cm) . The apparatus detects automatically all the mouse movements, and discriminates between total and ambulatory activity. The locomotor activity (number of movements across the beams) was counted during 5 min. An increase in the number of transitions through the beams reflects an augmented locomotor activity. (d) Horizontal Wire Test: It consisted of an horizontally strung wire (1mm diameter, 15cm long) , placed at 20cm from the table. Mice were lifted by the tail, allowed to grasp the wire with their forepaws and released. The number of mice that did not grasp the wire with their forepaws or actively grasped the wire with at least one hindpaw within 3 sec was determined. After two trials, performed at 5min. intervals, the test took place. A yorelaxant drug, like diazepam at high doses, will impair the ability of mice to grasp the wire. Generally, this state of muscle relaxation is commonly associated with sedation. General Experimental Procedure The general procedure for all the tests is as follows: mice were injected with vehicle or the drug solution 20min before the beginning of the test and put into another home cage. Ro 15-1788, a specific BZD receptor antagonist was injected lOmin before the tested drug. All the injections were given intraperitoneally (i.p.). Control mice were tested in each session, in parallel with those animals receiving the test drug or diazepam. Testing was carried out 'blind'. All data were submitted to analysis of variance (ANOVA) . Post-hoc comparisons between individual treatments and controls were made using Dunnett's t-test. Results The results of experiments in devices (a) to (d) are summarised in the Figures 1-18. Figure 1 shows ambulatory locomotor activity counts during a 5min test session in an OPTO-Varimex apparatus, 20min after IP injection with DZ (0.3-3 mg/kg), or chrysin (CHRY, 0.6-10mg/kg) . Data are expressed as medians (interquartile range) of (n) number of animals. *p< 0.05, **p< 0.02, ***p< 0.002 significantly different from controls (Mann Whitney test) . Figure 2 shows mean (±S.E.M.) percentage of open arm entries (hatched bars) and percentage of time (sec) spent in the open arms (closed bars) in mice given a 5min test in the elevated plus-maze, 20 min after i.p. injection with DZ (0.3 and 0.6 mg/kg), or CHRY (0.1-10 mg/kg). *p< 0.01, significantly different from controls (two-tailed Dunnett's t-test after analysis of variance) . Figure 3 shows mean (±S.E.M.) percentage of open arm entries (hatched bars) and percentage of time (sec) spent in the open arms (closed bars) in mice given a 5min test in the elevated plus-maze, 20min after i.p. injection with VEH, CHRY (lmg/kg) or CHRY + RO 15-1788 (3 mg/kg) administered i.p. lOmin before chrysin. * p< 0.01, significantly different from controls (two-tailed Dunnett's t-test after analysis of variance) . No significant differences were found in the total arm entries (F (2,49) = 3.18). Figure 4 shows mean (± S.E.M.) number of head-dips (closed bars) and time (sec) spent head-dipping (hatched bars) for mice given a 5min test in the holeboard, 20 min after an i.p. injection with DZ (0.3-6 mg/kg) or CHRY (1-10 mg/kg). *p< 0.05, **p< 0.01, significantly different from controls (two-tailed Dunnett's t-test after analysis of variance) . Figure 5 shows the performance of mice in the wire test 20min after an i.p. injection with DZ (3 and 6 mg/kg) or CHRY (0.6-30 mg/kg). The test took place after two trials, executed after a 5 min interval. Figure 6 shows mean (± S.E.M.) of total entries, percentage of open arm entries (% Nr open) and percentage of time (sec) spent in the open arms (% T open) in mice given a 5 min test in the elevated plus-maze, and ambulatory activity in -mice given a 5min test in an OPTO-VARIMEX apparatus, 20min after i.p. injection with vehicle and apigenin (1, 3, 10 mg/kg). Figure 7 shows mean (± S.E.M.) number of head-dips and time (sec) spent head-dipping and rearings for mice given a 5 min test in the holeboard, and performance to grasping the wire test, 20min after an i.p. injection with vehicle and apigenin 3 and 10 mg/kg; and Figure 8 shows noradrenaline levels in locus coeruleus nucleus after a session of immobilization stress alone or with pretreatments as indicated in the figure (noradrenaline is expressed as per cent of control value) Apigenin blocked almost completely the noradrenaline decrease provoked by stress - first bar. (Chrysin was almost equipotent with diazepam) . Figure 9 shows total entries, percentage of open arm entries (% number open) and percentage of time (sec) spent in the open arms (% time open) in mice given a 5 min test in the elevated plus-maze, and ambulatory activity in mice given a 5min test in an OPTO-VARIMEX apparatus, 20 min after i.p. injection with vehicle and flavone (1,3 mg/kg). Figures 10, 11, 12, 13 and 14 show mean (± S.E.M.) of total entries (hatched box) , percentage of open arm entries (closed box) and percentage cf time (sec) spent in the open arms (cross hatched box) in mice given a 5 min. test in the elevated plus-maze, 20 min. after i.p. injection with vehicle and a mixture of brominated flavone (0.6, 1,3 mg/kg) in Figure 10; 2' chlorinated chrysin (1,3 mg/kg) in Figure 11 and 2' fluorinated chrysin (3 mg/kg) in Figure 12; 6, 8 dibromo chrysin (lmg/kg) in Figure 13; 7-bromo flavone (0.5mg/kg) in Figure 14. * p<0.01, ** p<0.02, *** p<0.05, significantly different from the controls (student's t-test after analysis of variance) . Figures 15, 16, 17 and 18 show ambulatory locomoter activity counts during a 5 min. test session in an OPTO-Varimex apparatus, 20 min. after i.p. injection with a mixture of brominated flavones (0.6,1,3 mg/kg). Figure 15; 2' chlorinated chrysin (1,3 mg/kg); Figure 16; 2 ' fluorinated chrysin (3 mg/kg), Figure 17; 6,8 dibromoflavone, Figure 18. Data are expressed as medians (interquartile range) of (n) number of animals. No significant difference was observed in comparison to the controls (Mann Whitney test) . No significant change was observed in ambulatory activity when 7-bromo flavone was compared against a control (data not shown) . In all the experiments, diazepam DZ (0.3-6 mg/kg) was used as reference drug. Figure 1 shows the typical pharmacological profile of increasing locomotor activity by DZ. Similarly, there was a significant increase in locomotor activity with equipotent doses of chrysin (0.6-1 mg/kg) . Figure 2 shows the performance of mice following i.p. administration of vehicle, DZ or chrysin on the elevated plus maze. DZ (0.3 and 0.6 mg/kg) increased the percentage of entries in the open arms (p< 0.01) and the percentage of the time spent on the open arms (p< 0.01). Chrysin (1 mg/kg) produced also an increase in both parameters (p< 0.01). No differences were observed in the total arm entries (Table 1) . Thus both DZ and CHRY displayed an anxiolytic effect. The effect of chrysin (1 mg/kg) on the number of entries into and the time spent on the open arms was prevented by the prior administration of Ro 15-1788, a central BZD receptor antagonist (Figure 3). As shown in Figure 4 in the holeboard DZ (0.3 mg/kg) increased the number of head-dips (p< 0.05) and at 1 mg/kg increased the time spent head-dipping (p< 0.01). As expected, DZ (6 mg/kg) induced a decrease in both the number of head-dips and in the time spent head-dipping (p< 0.01) which indicates sedation. Chrysin (3 mg/kg) produced a significant increase in the time spent head-dipping but did not elicit sedative effects at high doses (10 mg/kg) . Figure 5 shows that at 6 mg/kg DZ significantly decreased the percentage of animals grasping the horizontal wire, indicating a muscle relaxant effect. On the other hand, chrysin (0.6-30 mg/kg) was ineffective in the same test and produced no muscle relaxant effect. Thus, chrysin had anxiolytic effects in the elevated plus maze test but did not exhibit sedation or muscle relaxation. Figures 6 and 7 show analogous results for apigenin (API.) Apigenin administered interperitonealy shows anxiolytic effects in the elevated plus maze test (Figure 6) ; but did not induce any effect in the holeboard test (Figure 7) or the horizontal wire test (Figure 7) indicating no sedative or muscle relaxant activity. Figure 8 shows that both chrysin and apigenin dampened the noradrenaline decrease in the locus coeruleus provoked by immobilisation stress. In comparison to diazepam, apigenin showed the most potent effect. The figure shows per cent change (related to controls) of locus coeruleus noradrenaline levels after stress and different treatment (as indicated in the figure) . Rats were submitted to a 90-minute immobilization stress session in a plastic cylinder. The locus coeruleus was dissected out after killing by decapitation and noradrenaline assessed by HPLC with electrochemical detection. Doses were lmg/kg for each substance, injected i.p., 30 minutes before the stress session. Figure 9 shows the performance of mice following i.p. administration of vehicle, D2 or flavone on the elevated plus-maze. Flavone (lmg/kg) increased the percentage of entries and time spent in the open arms (p<0.002) Mann-Whitney test. Figure 10 shows that at a concentration of 1 mg/kg a mixture of brominated flavones results in a significant increase in the number of entries into the open arms in comparison to the control. The corresponding ambulatory locomotion test (Figure 14) shows no significant decrease in activity. Thus, the increased entries in the elevated plus-maze test show a reduction in anxiety without a significant decrease in activity (i.e. without a significant sedative effect) . A similar effect can be seen for 1 mg/kg administered 2' chlorinated chrysin (Figures 11 & 16), for 3 mg/kg administered 2' fluorinated chrysin (Figures 12 & 17), for lmg/kg administered 6,8 dibromo chrysin (Figures 13 & 18) and for 0.5mg/kg administered 7-bromo flavone (Figure 14). EXAMPLE 3 (Competitive inhibition of H 3 -flunitrazepam to benzodiazepine receptor) This experiment was carried out as a general screen for compounds exhibiting benzodiazepine-like activity, in order to identify compounds for testing for specific anxiolytic activity. Binding of 3H-flunitrazepam (0.7nM) to the benzodiazepine receptor was carried out in extensively washed cerebral cortical membranes. H 3 -flunitrazepam has a Ki of 3 micromole to the benzodiazepine receptor. IC 50 values were obtained using flavonoids at different concentrations (10~ 10 to 10 ~3 M) . Table II shows the results of the tests. Flavone, chrysin, apigenin and 6,8 dibromochrysin (IC5o=0.7-3um) show benzodiazepine ligand behaviour similar to that of 3 H-flunitrazepam. 7-bromo flavone and amentaflavone show a far higher affinity for the benzodiazepine receptor (IC 5 o=0.05um and O.Olu respectively) than 3 H-flunitrazepam. The other flavonoids show weak benzodiazepine ligand behaviour. EXAMPLE 4 (Induced seizures in mice) Benzodiazepines in general show an anxiolytic effect, a sedative effect and an anticonvulsant effect. In order to assess the present compounds for anticonvulsant activity the following experiment was carried out. The method of Medina et al Biochem.Pharm. 4jD P2227-2231, (1990) was followed (with the exception that injections were carried out intraperitoneally) and the mice observed for seizures. Table III shows that apigenin has no anticonvulsant activity. This differs from the benzodiazepines and shows that the flavonoids have a more selective and specific mode of action, being limited to anxiolytic activity. TABLE I Total number of arm entries made by mice during 5 min test in the elevated plus-maze, 20 min after drug injection. DRUGS TOTAL (mg/kg) n ARM ENTRIES VEH (53) 8.7 ± 0.6 DZ 0.3 (21) 10.5 ± 0.8 0.6 (22) 12.4 ± 1.9 CHRYSIN 1 (36) 9.9 ± 0.8 3 (21) 8.7 ± 1.1 10 (15) 10.2 ± 1.1 Data are expressed as neans ± S.E.M. of n=number of animals. Analysis of variance F(5,160) = 2.27 " , p< 0.05 TABLE II Structure - activity relationships of several flavonoids on the 3 H Flunitrazepam binding to bovine brain membranes. FLAVONOID 3 H Flunitrazepam binding ICgnfuM) Flavone 1 Apigenin 3 Isoquercetin 10 6,8 dibromochrysin 0.7 Isoquercitrin 80 5 hydroxy 7 methoxyflavone 46 Rutin 60 2 ' fluorochrysin 8 2 ' chlorochrysin 9 chrysin 2 flavanone 40 7-bromoflavone 0.05 Amentoflavone 0.01 Ginkgetin 5 Isoginkgetin 4-5 TABLE III EFFECTS OF IP ADMINISTRATION OF APIGENIN ON PTZ INDUCED SEIZURES IN MICE. Doses (mg/kg) Number of animals with clonic convulsions PTZ 31/31 Diazepam 3 + PTZ 0/6 Apigenin 3 + PTZ 8/8 Apigenin 20 + PTZ 3/3 Apigenin 40 + PTZ 12/14 Apigenin 80 + PTZ 11/11 The convulsant doses of pentylenetetrazole are between 50-80 mg/kg in different experiments carried out in 6 independent days. 5,7-Dihvdroxy-2'-fluoroflavone(2'-fluorochrysin). Light tan prisms, mp 273-276°C. 'H NMR δ (DMSO- ,) 6.24 (lH.d, J = 1.8 Hz, H-6), 6.47 (lH,d, 7 = 1.8 Hz, H- 8), 6.69 (1H,J,H-3), 7.44 (2H.iw,H-3V-6'), 7.67 (lH.ώtø, J = 6.1, 7" = 1.3 Hz.H- 4' or -5'),7.98 (IH.ddd, 7 = 7 '= 7.4, J" = 1.2 Hz, H-5' or -4'), 12.67 (1H, s, C-5- OH). 13 C NMR δ (DMSO-tf 6 ) 94.29(C-8), 99.33(C-6), 104.05 (C-4a), 109.84 (d, 7 = 10.6 Hz. C-3), 117.10 (d, 7 = 21.9 Hz, C-6'), 119.38 (d, 7 = 10.6 Hz, C-l'), 125.42 (d, 7 = 3.8 Hz, C-3'), 129.72 (C-5' or -4'), 134.05 (d, J = 36 Hz, C-4' or - 5'), 157.72 (C-8a), 158.71 (d, 7 = 75 Hz, C-2'), 159.21 (C-2), 161.60(C-5), 164.82(C-7), 181.63(C-4). 5,7-Dihydroxy-2'-chloroflavone(2'-chlorochrysin). Yellow granularpowder, sublimes from 225°C, melts 273-275°C. Η NMR δ (OMSO-d 6 ) 6.35 (1H, d, 7 = 1.8 Hz, H- 6), 6.50 (1H, d, 7 = 1.8 Hz, H-8), 6.66(1H, s, H-3), 7.63 (1H, ddd, 7 = 7' = 7.5, J" = 1.3 Hz. H-5' or -4'), 7.70 (1H, ddd, 7 = 7' = 7.6, 7" = 1.3 Hz, H-4' or 5'), 7.76 (1H, dd, 7=7.8, 7' •= 1.0 Hz H-6' or -3'), 7.78 (1H, dd, 7 = 7.4, 7' = 1.2 Hz, H-3' or -6'), 12.67 (1H, s, C-5-OH). 13 C NMR δ (DMSO-</ 6 ) 94.35 (C-8), 99.54 (C-6), 103.96 (C-4a), 110.76 (C-3), 127.97 (C-5'or -4'), 130.69 (C-6' or 3'), 131.13 (C-l), 131.50 (C-3' or 6'). 131.81 (C-2'), 132.88 (C-4' or -5'), 158.02 (C- 8a), 161.75 (C-5), 162.86 (C-7), 165.26 (C-2), 181.58 (C-4). 6,8-Dibromo-5, 7-dihydroxyflavone (6, 8-dibromochrysin). Prepared by bromination of chrysin at room temperature with excess bromine in acetic acid. Very fine, light yellow * needles, subliming from 265 °C to give prisms, mp (309) 320°C, 1H NMR δ (DMSO-</ 6 ) 7.17 (s, H-3), 7.59 (dd, 7 = 7' = 6.5Hz. H-375'), 7.62 (dd, J = 7' = 6.5 Hz, H-4'), 8.12 (d, J = 6.4 Hz, H-276'), 13.71 (s, C-5-OH). 13 C NMR δ (DMSO- ,) 88.37 (C-8), 94.47 (C-6), 105.03 (C-4a), 126.0 (C-3), 126.37 (C-276'), 129.14 (C-375'), 130.13 (C-l '), 132.35 (C-4'), 152.18 (C-7), 156.97 (C-8a or -5), 157.34 (C-5 or 8a), 163.41 (C-2). 181.44 (C-4). Tectochrysin (5-hydroxy-7-methoxyflavone). Prepared from chrysin by warming with one equivalent of dimethyl sulphate in DMF with finely ground K,CO 3 . Fine, light yellow needles, mp 175-180 °C (lit. 163°C). 'H NMR δ (OMSO-d 6 ) 3.87 (-s,OCH 3 ), 6.39 (d, 7 = 1.7 Hz, H-6), 6.80 (d, 7= 1.7 Hz, H-8). 7.02 (s,H-3), 7.58 (dd, 7=7'= 7.8 Hz. H-375'), 7.60 (rt, 7 « 7.8 Hz, H-4'), 8.09 (d,J = 7.8 Hz, H-276'), 12.80 (s, C-5-OH). 5,7-Dimethoxyflavone (chrysin 5,7-di-O-methyl ether). Prepared similarly to the previous compound, but with excess dimethyl sulphate. Light tan powder, mp 147- 149 °C. 'H NMR δ (CDC1 3 ) 3.91 (s,OCH-), 3.96 (s,OC -), 6.37 (d, 7=2.0 Hz, H- 6), 6.57 (d, 7 = 2.2 Hz, H-8), 6.68 (s, H-3), 7.50 (dd, H-375'), 7.51 (tt, H-4'), 7.87 (d, 7 = 5.3 Hz?, H-276'). 2'-Chloro-5-hydroxy-7-methoxyflavone (2'-chlorotectochrysin). Prepared from 2'- chlorochrysin by warming with one equivalent of dimethyl sulphate in DMF with finely ground K 2 CO 3 . Light yellow needles, sublimes from 160°C, melts 183-185°C. Η NMR δ (DMSO- ,). 3.88 CΪ,OCH 3 ), 6.47 (br s, H-6), 6,66 (s, H-3), 6.72 (br s, H-8), 7.6 (m, H-37475'), 7.82 (br d, 7 = 6.9 Hz, H-6'), 12.64 (s, C-5-OH). 2'-Chloro-6,8-dibromo-5,7-dihydroxyflavone (2 '-chloro-6, 8-dibromochrysin). Prepared from 2 '-chlorochrysin by bromination with excess bromine in acetic acid. Very fine, pale yellow needles, sublimes from 250°C, melts 285-290°C. 'H NMR δ (DMSOO 6.83 (s, H-3), 7.6 (3H, m, H-37475'), 7.84 (d, 7=6.2 Hz, H-6'), 13.59 (s, C-5-OH). 13 C NMR δ DMSO-d 6 ) 88.66 (C-8), 94.97 (C-6) 105.20 (C-4a), 111.02 (C-3), 128.02 (C-5'), 130.50 (C-6'), 130.90 (C-l'), 131.72 (C-3'), 131.91 (C-4'), 133.24 (C-2'), 152.99 (C-7), 157.26 (C-8a or -5), 157.88 (C-5 or - 8a), 163.30 (C-2), 181.40 (C-4).

Claims: CLAIMS 1. A method of treatment of anxiety in a patient which comprises administering to the patient an effective non-toxic amount of a flavonoid compound of general formula wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 8 are independently selected from H, OH, R, NO2, halo, OR, NH2, NHR, NR 2 , COOR, COOH, CN or a sugar group; R 6 , and R 7 are both H, or R 6 and R 7 together form a single bond; R is Cι_ 6 alkyl or alkenyl; or the administration of " an effective non-toxic amount of a biflavonoid which is a dimer of a compound of general formula (I) . 2. A method according to claim 1 wherein R 6 and R 7 are both H. 3. A method according to claim 1 wherein R 6 and R 7 together form a single bond. 4. A method according to claim 3 wherein R 1 , R 2 , R 3 , R 4 and R 5 are independently selected from H, OH, and halo. 5. A method according to claim 4 wherein R 1 and R 3 are both hydroxy. 6. A method according to claim 5 wherein the compound of general formula (I) is chrysin. 7. A method according to claim 4 wherein R 1 , R 3 and R 5 are all hydroxy. 8. A method according to claim 7 wherein the compound of general formula (I) is apigenin. 9. A method according to claim 4 wherein R 5 is selected from OH and halo. 10. A method according to claim 5 wherein R 5 is halo. 11. A method according to claim 10 wherein the compound of general formula (I) is selected from 2'-chlorochrysin and 2'-fluorochrysin. 12. A method according to claim 5 wherein R 2 and R 4 are both halo. 13. A method according to claim 12 wherein the compound of general formula (I) is 6,8-dibromochrysin. 14. A method according to claim 5 wherein R 3 is halo. 15. A method according to claim 14 wherein the compound of general formula (I) is 7-bromochrysin. 16. A method according to claim 1 wherein the biflavonoid dimer has the general formula (II) . wherein; R 1 to R 8 and R have the meanings given in claim 1. 17. A method according to claim 16 wherein R 1 , R 3 and R 5 in each of the dimer moieties of general formula (I) are hydroxy or methoxy. 18. A method according to claim 17 wherein the biflavonoid dimer is amentoflavone, ginkgetin or isoginkgetin. 19. A method according to any preceding claim wherein the treatment reduces anxiety without exerting any substantial sedative effect. 20. A pharmaceutical formulation which comprises a flavonoid compound of general formula (I) as set out in claim 1 or a dimer thereof in admixture with a pharmaceutically acceptable carrier; with the proviso that R 1 to R 8 are not all H; R 1 and R 3 are not both OH when R 2 and R 4 to R 8 are H; and R 1 and R 3 are not both OH and R 5 is not 4'hydroxy, when R 4 and R 6 to R 8 are H. 21. A formulation according to claim 20 wherein R 1 , R 2 , R 3 , R 4 and R 5 are independently selected from H, OH and halo. 22. A formulation according to claim 21 wherein R 3 is halo, and R 1 , R 2 and R 4 ar.d R 5 are H. 23. A formulation according to claim 21 wherein R 5 is halo, and R 1 and R 3 are both OH. 24. A formulation according to claim 23 wherein the compound of general formula (I) is 2'-chlorochrysin or 2'-fluorochrysin. 25. A formulation according to claim 21 wherein R 2 and R 4 are both halo. 26. A formulation according to claim 25 wherein the compound of general formula (I) is 6,8-dibromochrysin. 27. A formulation according to claim 21 wherein R 3 is halo. 28. A formulation according to claim 27 wherein the compound of general formula (I) is 7-bromochrysin. 29. A formulation according to claim 20 wherein the biflavonoid dimer has ne general formula (II) . wherein; R 1 to R 8 and R have the meanings given in claim 20, 30. A formulation according to claim 29 wherein R 1 , R 3 and R 5 in each of the dimer moieties of general formula (I) are hydroxy or methoxy. 31. A formulation according to claim 30 wherein the biflavonoid dimer is amentoflavone, ginkgetin or isoginkgetin. 32. A flavono wherein R selected from H, OH and halo. R 6 and R 7 are both H, or R 6 and R 7 together form a single bond; and R is Cι_ 6 alkyl or alkenyl. with the proviso that at least one of R 1 , R , R 3 , R 4 and R 5 is halo. 33. A flavonoid compound according to claim 32 which is selected from 2'-chlorochrysin 2'-fluorochrysin 6,8-dibromochrysin, and 7-bromoflavone. 34. The use of a flavonoid compound of general formula (I) as defined in claim 1 or a biflavonoid which is a dimer thereof for the preparation of a medicament for the treatment of anxiety in a patient.

Inventors: CASSELS BRUCE KENNEDY, DAJAS FEDERICO JOSE, MEDINA JORGE HORACIO, PALADINI ALEJANDRO CONSTANTINO, SILVEIRA RODOLFO HORACIO, CASSELS, BRUCE, KENNEDY, DAJAS, FEDERICO JOSE, MEDINA, JORGE, HORACIO, PALADINI, ALEJANDRO, CONSTANTINO, SILVEIRA, RODOLFO, HORACIO
Applicants: CASSELS BRUCE KENNEDY, DAJAS FEDERICO JOSE, MEDINA JORGE HORACIO, PALADINI ALEJANDRO CONSTANTINO, SILVEIRA RODOLFO HORACIO, UNIV STRATHCLYDE, CASSELS, BRUCE, KENNEDY, DAJAS, FEDERICO JOSE, MEDINA, JORGE, HORACIO, PALADINI, ALEJANDRO, CONSTANTINO, SILVEIRA, RODOLFO, HORACIO, THE UNIVERSITY OF STRATHCLYDE
Application year: 1994
Main classification: A61K31
IPC codes: A61K31, A61P25, C07D311